The broad objective of this renewal is to increase our understanding of the workings of the complement system in causing human disease. The long-term goal is to define inflammatory pathways mediating tissue injury in order to target specific therapeutic agents to prevent or halt the undesirable reaction. Specifically, mutations have been identified in regulatory proteins of the alternative pathway (AP) in atypical hemolytic uremic syndrome (aHUS) and, more recently, in preeclampsia (PE). These risk variants lead to what has been called the hyperinflammatory complement phenotype or the complement inflammasome. For a given degree of cellular injury, in these individuals there is excessive activation of the AP resulting in tissue injury and disease states, primarily related to thrombotic lesions in the microvasculature (thrombomicroangiopathy). A large part of this grant is focused on CD46, the complement inhibitory protein that is widely expressed on cell membranes. In the first Specific Aim, we will characterize how CD46 mutations identified in aHUS and PE alter regulatory activity. In part b of this first aim, we will employ the new technology of targeted resequencing of multiple genes implicated in several large cohorts of patients with PE, lupus, antiphospholipid syndrome, and aHUS. Complement and clotting genes will initially receive the most attention. In Specific Aim II, we will explore the membrane behavior and signaling capabilities of CD46 in multiple cell types including T lymphocytes, epithelial cells and endothelial cells. On many cells, CD46 is down-modulated by ligand engagement and by cross-linking with antibodies. The process by which this is accomplished will be characterized and compared. Proteolytic processing and vesicle formation will be monitored in each cell population to determine their contribution to shedding and signaling. We also have strong evidence for a specific kinase interacting with the cytoplasmic tails of CD46. We will address many unanswered questions about CD46's membrane behavior including shedding, signaling capability, interactions with seven human pathogens and promotion of T regulatory cell development. While CD46's complement inhibitory profile is established, its role in these more recently described phenomena is largely enigmatic.